請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21042
標題: 蟑螂過敏原p72在融合瘤細胞中的表現
Expression of cockroach allergen p72 in hybridoma
作者: 李瑞岳
Lee, Rueih-Yueh
關鍵字: hybridoma
融合瘤細胞
cockroach
allergen
expression
蟑螂
過敏原
表現
出版社: 分子生物研究所
摘要: Cockroach is one of the major indoor causative agents for inducing of allergic bronchial asthma. Dr. C.-H. Wu at Taichung Veterans General Hospital, Taichung, Taiwan found that there are at least 18 allergens in the whole body extract of American cockroach. One of the major allergens is a 72-kDa polypeptide (designated as p72 in this study). The p72 cDNA (clone C20) was identified from a cDNA expression library of American cockroach and expressed in E. coli. Unfortunately, the p72 from E. coli is insolubleeven after denaturation and refolding processes in vitro. Sequence analysis revealed that p72 is possibly a glycoprotein with two potential N-glycosylation sites Asn-Phe- Thr and Asn-Thr-Thr at aa 142 and aa 177, respectively. Therefore, the insolubility of the p72 expressed in E. coli may have resulted from the lack of glycosylation. To solve this problem, a hybridoma-based expression system was developed to express the p72 protein. Plasmid pg2bVH was a derivative of pSV2 gpt carrying an insert cloned fromthe genomic DNA of hybridoma 36-65 which contained the promoter, leader (lH) exon , and VDJ exon followed by the IG heavy (H) chain enhancer (EH) and mouse g2b constant (Cg2b)-region gene. This plasmid has been shown to express g2b heavy chain efficiently. To construct a p72- expression plasmid, the p72 coding region in the cDNA clone C20 was amplified by PCR, then the fragment obtained was cloned into pg2bVH to replace the VDJ exon. The resulting plasmid, pg2b-p72, was introduced into Sp2/0-Ag14 cells byelectroporation, and the p72-production ability of tansfectomas was examined by Western blot analysis. Among hundreds, only one clone, designated as Sp2/0-p72 secreted p72 transiently, even though the plasmid DNA was integrated into the genome. Presence of the p72 DNA in the most clones was verified by PCR amplification on the genomic DNA of the transfectomas using sequences within the p72 coding region as primers. However, Southern blot analysis using the plasmid pg2b-p72, pSV2gpt, and p72 DNA as probesshowed that only Sp2/0-p72 possesses an intact transcription unit of recombinant p72 gene. This may explain the low p72-expression efficiency in transfectomas. Nevertheless, the loss of p72 expression in Sp2/0-p72 remained to be determined.
蟑螂為引起過敏的一個主要室內來源之一。台中榮總吳啟輝博士發現美國 蟑螂萃取液中含有至少 18 種致敏成分,其主要的致敏原之一為分子量為 72kDa 之蛋白 (稱為p72)。以抗體由蟑螂的 cDNA 表現庫篩選出 p72 的 cDNA (C20),進行核酸定序及表現;結果由 Escherichia coli (E. coli) 生產的重組蛋白為不可溶的。由 cDNA 推衍出胺基酸序列而估算分 子量,推測 p72 為一種醣蛋白。由於醣化可能影響蛋白質的溶解性,然 而E. coli卻缺乏醣化機制;此可能是造成重組 p72 不溶的原因。本研究 嘗試以融合瘤細胞來表現 p72,希望能找到一個適於生產 p72 的表現系 統。表現質體 pg2bVH 在融合瘤細胞中能非常有效的表現免疫球蛋白之重 鏈 (IgH)。實驗首先以 PCR 將含有 p72 基因的 DNA 片段由 C20 上增幅 後,取代質體 pg2bVH 上 IgH 基因的 VDJ exon 片段成為 pg2b-p72。再 用電孔法將表現載體送入融合瘤細胞,以 western blotting 分析各轉化 細胞表現 p72 之能力。結果由百餘株轉化細胞株中,僅篩選到一株能短 暫表現且分泌 p72 的 Sp2/0 轉殖株,稱為 Sp2/0-p72。為了探究轉化作 用的低效率情形,設計以 p72 基因中序列為引子對,轉化細胞的染色體 DNA 為模板,進行 PCR;結果發現在 8 株隨機挑選的細胞株中,7 株有 預期大小的 DNA 片段產生,證明質體 DNA 已嵌入染色體基因組中。再分 別以整個表現質體 pg2b-p72、含有pSV2gpt的DNA片段,以及 p72 基因片 段為模本製作探針,進行 southern blot 分析。發現只有在 Sp2/0p-p72 的樣品中有相同的 DNA 片段 (約 13 及14kb) 為三探針所偵測到;其大 小大於免疫球蛋白重鏈基因啟動子所控制的轉錄區 (transcription unit)(約11.8kb)。其它各樣品中,與 pg2b-p72 及 p72 DNA 探針反應的 DNA 片段並不相同,且與p72 DNA有反應的 DNA 片段均未大於 11.8kb; 而 gpt 探針所偵測到的 DNA 片段均大於其轉錄區。此結果顯示大半質體 在嵌入染色體時,可能使含有 p72 的轉錄區被中段,因此無法表現p72蛋 白,但仍能於篩選培養液中存活。至於 Sp2/0-p72 含有完整 p72 轉錄區 ,卻失去表現 p72 蛋白的原因,則有待進一步之探討研究。
URI: http://hdl.handle.net/11455/21042
顯示於類別:分子生物學研究所

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